Method of treating a cancer patient without the need for a tissue biopsy

ABSTRACT

Provided herein, among other things, is a method of treating a cancer patient without the need for a tissue biopsy. In some embodiments, the method may comprise (a) performing or having performed a sequencing assay on cell-free DNA (cfDNA) from a sample of blood from the patient to determine if the cell-free DNA comprises actionable and/or non-actionable sequence variations in one or more target genes, and (b) treating the patient using the following method: i. administering a therapy that is targeted to an actionable sequence variation if the patient is identified as having the actionable sequence variation, and ii. administering a non-targeted therapy in the absence of any follow-up genetic testing on DNA extracted from a tissue biopsy if one or more non-actionable sequence variations and no actionable sequence variations are identified.

CROSS-REFERENCING

This application claims the benefit of U.S. provisional application Ser.No. 62/727,462, filed on Sep. 5, 2018, which application is incorporatedby reference herein.

BACKGROUND

Non-small cell lung cancer (NSCLC) accounts for over 85% of lung cancer[1], and the majority of patients present with advanced stage diseaseand are treated with systemic therapies. Great strides have been made inthe development of therapies for such patients, including targetedtherapies and immunotherapy. Targeted therapies require identificationof specific molecular alterations in the cancer [2] and guidelinesrecommend broad genomic profiling to assess for therapeutic targets.However, the utilization of such comprehensive testing is still limited,often due to inadequate tumor tissue in many patients given the hightissue demands of comprehensive genomic profiling (CGP) testing. Arecent review of over 800 patients from routine US community oncologypractices revealed that only 59% of patients were profiled for two ofthe best known genomic alterations (EGFR mutations and ALK fusions), andonly 8% received CGP covering all the recommended alterations [3].Repeat biopsies are costly, often result in patient discomfort, and manypatients may experience complications [4]. A recent US Medicare basedanalysis demonstrated that the average cost of a transthoracic biopsywas $14,587 once treatment of complications was included [5].

Plasma based assays for molecular profiling of tumor mutations throughsequencing of cell-free (cfDNA) offer the potential to overcomedifficulties associated with tissue based CGP. These less-invasive“liquid biopsies” are now entering routine clinical practice, withrecent National Comprehensive Cancer Network (NCCN) guidelinesrecommending their use in NSCLC patients when tissue biopsy is notavailable [6].

Cancers that are associated with actionable mutations typically have abetter prognosis than other cancers because they can be treated withtherapies that specifically target an activity of the protein having thevariation. It is common to identify no actionable mutations in cell-freeDNA, however. In these cases, it is often unclear whether the tumoritself is not associated with an actionable mutation or the number ofmolecules that have an actionable mutation in the cfDNA is below thedetection limit of the assay. In these cases, a follow-up tissue biopsyis often performed in order to confirm the results obtained from cfDNA.

Follow-up tissue biopsies are expensive, frequently result in patientdiscomfort, and can cause complications. As such, any methods that allowtreatment decisions to be made using data obtained from sequencing cfDNAalone, i.e., without performing a tissue biopsy, have significant value.

SUMMARY

Some embodiments of the present method are based, at least in part, onthe discovery of a correlation between the identification ofnon-actionable sequence variations in cfDNA from a patient and lack ofactionable sequence variations in a tissue biopsy from the same patient,if no actionable sequence variations are found in the cfDNA. Thiscorrelation may be practically applied to make treatment decisionswithout having to perform a follow-up biopsy.

Provided herein, among other things, is a method of treating a cancerpatient without the need for a tissue biopsy. In some embodiments, themethod may comprise (a) performing or having performed a sequencingassay on cell-free DNA (cfDNA) from a sample of blood from the patientto determine if the cell-free DNA comprises actionable and/ornon-actionable sequence variations in one or more target genes, and (b)treating the patient using the following method: i. administering atherapy that is targeted to an actionable sequence variation if thepatient is identified as having the actionable sequence variation, andii. administering a non-targeted therapy in the absence of any follow-upgenetic testing on DNA extracted from a tissue biopsy if one or morenon-actionable sequence variations and no actionable sequence variationsare identified.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 shows two flow charts A and B. Flow chart A illustrates thestandard approach for making treatment decisions, which are based onactionable actionable changes only. Some of the principles of thepresent “rule out” method are illustrated in flow chart B.

FIG. 2 is a flow chart illustrating how the present method can be usedto avoid tissue biopsies in the treatment of non-small cell lung cancer.

FIG. 3 is a flow chart illustrating a second embodiment of the presentmethod that can be used to avoid tissue biopsies in the treatment ofnon-small cell lung cancer. This embodiment uses the allele frequency ofnon-actionable changes to determine the probability that actionablechanges could have been missed.

FIG. 4 is a flow chart illustrating how the present approach could beused to make treatment decisions for colorectal cancer patients.

FIG. 5 shows concordance data for clinically relevant alterationsdetected in the 8 key genes (ERBB2, ALK, ROS1, BRAF, MET, EGFR, STK11and KRAS) when both tissue and ctDNA testing was successful.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have at least 10, at least 100, at least 100, at least 10,000, atleast 100,000, at least 10⁶, at least 10⁷, at least 10⁸ or at least 10⁹or more members.

The term “sequencing,” as used herein, refers to a method by which theidentity of at least 10 consecutive nucleotides (e.g., the identity ofat least 20, at least 50, at least 100 or at least 200 or moreconsecutive nucleotides) of a polynucleotide is obtained.

The terms “next-generation sequencing” or “high-throughput sequencing”,as used herein, refer to the so-called parallelizedsequencing-by-synthesis or sequencing-by-ligation platforms currentlyemployed by Illumina, Life Technologies, and Roche, etc. Next-generationsequencing methods may also include nanopore sequencing methods such asthat commercialized by Oxford Nanopore Technologies,electronic-detection based methods such as Ion Torrent technologycommercialized by Life Technologies, or single-moleculefluorescence-based methods such as that commercialized by PacificBiosciences.

The term “sequencing at least part of the coding sequences” refers tosequencing at least 20% of, at least 40% of, at least 60% of, at least80% of, or at least 90% of (e.g., all of), of the coding sequences.

The term “reference sequence”, as used herein, refers to a knownnucleotide sequence, e.g. a chromosomal region whose sequence isdeposited at NCBI's Genbank database or other databases, for example. Areference sequence can be a wild type sequence.

As used herein, the terms “cell-free DNA from the bloodstream”“circulating cell-free DNA” and “cell-free DNA” (“cfDNA”) refers to DNAthat is circulating in the peripheral blood of a patient. The DNAmolecules in cell-free DNA may have a median size that is below 1 kb(e.g., in the range of 50 bp to 500 bp, 80 bp to 400 bp, or 100-1,000bp), although fragments having a median size outside of this range maybe present. Cell-free DNA may contain circulating tumor DNA (ctDNA),i.e., tumor DNA circulating freely in the blood of a cancer patient orcirculating fetal DNA (if the subject is a pregnant female). cfDNA canbe obtained by centrifuging whole blood to remove all cells, and thenisolating the DNA from the remaining plasma or serum. Such methods arewell known (see, e.g., Lo et al, Am J Hum Genet 1998; 62:768-75).Circulating cell-free DNA can be double-stranded or single-stranded.This term is intended to encompass free DNA molecules that arecirculating in the bloodstream as well as DNA molecules that are presentin extra-cellular vesicles (such as exosomes) that are circulating inthe bloodstream.

As used herein, the term “circulating tumor DNA” (or “ctDNA”) istumor-derived DNA that is circulating in the peripheral blood of apatient. ctDNA is of tumor origin and originates directly from the tumoror from circulating tumor cells (CTCs), which are viable, intact tumorcells that shed from primary tumors and enter the bloodstream orlymphatic system. The precise mechanism of ctDNA release is unclear,although it is postulated to involve apoptosis and necrosis from dyingcells, or active release from viable tumor cells. ctDNA can be highlyfragmented and in some cases can have a mean fragment size about 100-250bp, e.g., 150 to 200 bp long. The amount of ctDNA in a sample ofcirculating cell-free DNA isolated from a cancer patient varies greatly:typical samples contain less than 10% ctDNA, although many samples haveless than 1% ctDNA and some samples have over 10% ctDNA. Molecules ofctDNA can be often identified because they contain tumorigenicmutations.

As used herein, the terms “treat”, “treatment” and “treating” or thelike herein refers to administering a compound or pharmaceuticalcomposition as provided herein for therapeutic purposes. A treatmentinvolves administering treatment to a patient already suffering from adisease thus causing a therapeutically beneficial effect, such asameliorating existing symptoms, ameliorating the underlying metaboliccauses of symptoms, postponing or preventing the further development ofa disorder, and/or reducing the severity of symptoms that will or areexpected to develop.

As used herein, the term “therapeutically effective amount” refers tothe amount of a compound that, when administered to a patient having adisease, is sufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the subjectto be treated.

As used herein, the term “tissue biopsy” refers to a sample of canceroustissue taken from the body in order to examine it more closely. A biopsymay be from bone marrow, skin or from an internal organ and may becollected by an endoscopic biopsy (e.g., cystoscopy, bronchoscopy orcolonoscopy), a fine needle aspiration, a core needle, or surgery forexample.

As used herein, the term “actionable sequence variation” is a sequencevariation for which there is a therapy that specifically targets theactivity of the protein having the variation. In many embodiments anactionable sequence variation causes an increase in an activity of theprotein, thereby resulting in cells containing the variation to grow,divide and/or metastasize without check and in combination with othervariations, such as in tumour suppressor genes, leading to cancer.

As used herein, the term “therapy that is targeted to an actionablesequence variation” is a therapy that targets the activity of theprotein having the sequence variation. Therapy that is targeted to anactionable sequence variation often inhibits an activity of the mutatedprotein. Examples of actionable sequence variations for non-small celllung cancer and some other cancers, as well as therapies that targetthose actionable variations, are listed below.

As used herein, the term “non-actionable sequence variation” is asequence variation for which there is no therapy that is specificallytargeted to the activity of the protein having the variation.

As used herein, the term “non-targeted therapy” is a therapy that is nottargeted to a particular sequence variation. Non-targeted therapiesinclude radiation therapy, systemic or local chemotherapy, hormonetherapy, immunotherapy (e.g., immune checkpoint inhibition) and surgery.Examples of systemic chemotherapies for non-small cell lung cancer andsome other cancers include platinum based doublet chemotherapy such asthe combination of cisplatin and pemetrexed and the combination ofcisplatin and gemcitabine.

As used herein, the term “mutually exclusive” refers to sequencevariations that tend not to occur together in the same patient. Somaticmutations in the pathways that drive cancer development tend to bemutually exclusive across tumors. Such variations are identified throughanalysis of hundreds of tumor samples. See, for example, Cisowski et al(Small GTPases 2017 8: 187-192) and Mark et al (Bioinformatics 2016 32:i736-i745). For example, KRASG12D and BRAFV600E are mutually exclusivein lung, colorectal and other cancers (see, e.g., Sparks Cancer Res 58:1130-1134 and Cisowski, supra), although many others are known.

As used herein, the terms “genetic test” and “genetic testing” refer toany assay that is capable of detecting sequence variations (e.g.,mutations) in DNA or RNA. A genetic test may be done by sequencing,microarray, multiplex ligation-dependent probe amplification, Invaderassay, primer extension, ligation, or PCR such as ARMS-PCR or real timePCR, for example.

Other definitions of terms may appear throughout the specification.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, the someexemplary methods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Provided herein is a method of treating a cancer patient without theneed for a tissue biopsy. In some embodiments, the method may comprise:(a) performing or having performed a sequencing assay on cell-free DNA(cfDNA) from a sample of blood from the patient to determine if thecell-free DNA comprises actionable and/or non-actionable sequencevariations in one or more target genes, and (b) treating the patient byi. administering a therapy that is targeted to an actionable sequencevariation if the patient is identified as having the actionable sequencevariation, and ii. administering a non-targeted therapy in the absenceof any follow-up genetic testing on DNA extracted from a tissue biopsyif one or more non-actionable sequence variations and no actionablesequence variations are identified.

For non-small cell lung cancer, actionable variants are most commonlyfound in EGFR, ALK, ROS1, and BRAF, where the actionable variations inEGFR are activating mutations, the actionable variations in ALK includeALK gene fusions, the actionable variations in ROS1 include ROS1 genefusions; and the actionable variations in BRAF are activating mutations.Tumors that harbor activating genomic alterations in the correspondingkinase region of genes including EGFR and BRAF that result inconstitutive activation and have been identified as driver mutations(see, e.g., Gridelli et al, Nat Rev Dis Prim. 2015, which isincorporated by reference herein). Likewise, chromosomal rearrangementsbetween both ALK and ROS1 and fusion partners, have been identified asdrivers. This results from either ALK or ROS1's kinase domain being putunder the control of a new promoter. Targeted therapies directed againstthese activating alterations in EGFR, ALK, ROS1 and BRAF have beenapproved for use in patients harboring these activating mutations andfusions, and thus, these are described as “actionable” mutations. Assuch, in some embodiments, the target genes assessed for actionablesequence variations comprise EGFR, ALK, ROS1, and BRAF. For example, insome embodiment the method may further comprise sequencing at least partof the coding sequences of EGFR and BRAF and determining whether thereare any rearrangements in ALK and ROS1 that would result in theproduction of a fusion protein.

Table 1 below shows examples of actionable variations in EGFR, ALK, ROS1and BRAF as well as therapies that are targeted to cancers, e.g.,non-small cell lung cancer, that are associated with those actionablesequence variations. These therapies should not be limited to non-smallcell lung cancer because the variations can be found in a variety ofdifferent cancer types. Many other actionable sequence variations areknown.

TABLE 1 Actionable variations and therapies targeted to those variationsActionable sequence Gene variation Targeted therapy EGFR L858R, exon 19EGFR tyrosine kinase inhibitor (TKI) deletion, L861Q, therapy usinge.g., erlotinib (Tarceva), G719X, p.S768I, afatinib (Gilotrif),gefitinib (Iressa) or V765A, T783A, osimertinib (Tagrisso). V774A,S784P, and L861X ALK EML4-ALK, STRN- ALK tyrosine kinase inhibitor (TKI)ALK, KIF5B-ALK, therapy using, e.g., crizotinib (Xalkori), and TFG-ALKceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig). ROS1CD74-ROS1, ROS1 tyrosine kinase inhibitor (TKI) SLC34A2-ROS1, therapyusing, e.g., crizotinib (Xalkori), SDC4-ROS1, entrectinib (RXDX-101),lorlatinib (PF- TPM3-ROS1, 06463922), crizotinib (Xalkori), and EZR-ROS1entrectinib (RXDX-101), lorlatinib (PF- 06463922), ropotrectinib(TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib (Cometriq,Cabometyx). BRAF V600E, L601G, BRAF inhibitor therapy using, e.g.,K601E, L597V/Q/R emurafenib (Zelboraf), dabrafenib and G469V/S/R/E/A(Tafinlar), and encorafenib (Braftovi) or trametinib (Mekinist)

In some embodiments, the target genes assessed for actionable sequencevariations also include MET, RET and HER2. Actionable sequencevariations in MET include high-level MET amplification or MET exon 14skipping mutation, the targeted treatment for which include, TKItherapy, e.g., crizotinib (Xalkori). Actionable sequence variations inRET include RET fusions, including KIF5B-RET, TRIM33-RET, CCDC6-RET,NCO4A-RET fusions, the targeted treatment for which include cabozantinib(Cometriq, Cabometyx) and vandetanib (Caprelsa). Actionable sequencevariations in HER2 (ERBB2) include HER2 Exon 20 insertion, the targetedtreatment for which include ado-trastuzumab emtansine (Kadcyla). In someembodiments, two or more mutations may be only be clinically actionablewhen present together.

BRAF is the human gene that encodes a protein called B-Raf. The gene isalso referred to as proto-oncogene B-Raf and v-Raf murine sarcoma viraloncogene homolog B, while the protein is more formally known asserine/threonine-protein kinase B-Raf. The BRAF gene is located onchromosome 7q34, and covers approximately 190 kb. It contains at least19 exons and encodes a full-length transcript of 2,510 bp (NM_00433). Atleast seven variant transcripts have been identified, which are productsof alternative splicing. From these various transcripts, severalproteins are translated, including the full-length, 94-95 kD, 783 aminoacid product. See, e.g., Sithanandam et al, Oncogene 1990 5: 1775-80;and Meyer et al Journal of Carcinogenesis 2003 2, 7. The sequence ofhuman BRAF and its structure are set forth in entry 673 in NCBI's genedatabase; NCBI Reference Sequence: NG_007873.3.

EGFR encodes a transmembrane glycoprotein that is a member of theprotein kinase superfamily. The gene maps to 7p11.2. The EGFR genecontains 28 exons and spans nearly 200 kb. Intron 1 spans 123 kb. Thegene contains several repeat elements, including SINEs and LINEs, aswell as a trinucleotide (TGG/A) repeat-rich region in intron 15, and 2long CA repeats in intron 27. The sequence of the human EGFR gene andits structure are set forth in entry 1956 in NCBI's gene database; NCBIReference Sequence: NG_007726.3. See, e.g., Zhang et al J Med Genet.2007 44: 166-72.

ALK encodes a receptor tyrosine kinase, which belongs to the insulinreceptor superfamily. ALK is situated on the short arm of chromosome 2(2p23.2). The gene contains over 30 distinct introns and transcriptionproduces about 8 different mRNAs, with several alternatively splicedvariants and unspliced forms. The sequence of the human ALK gene and itsstructure are set forth in entry 427 in NCBI's gene database; NCBIReference Sequence: NC_000002.12. See, e.g., Figueiredo-Pontes et al JThorac Oncol. 2014 February; 9(2): 248-253.

ROS1 encodes a receptor tyrosine kinase with structural similarity tothe ALK protein; it is encoded by the c-ros oncogene, which is found onChromosome 6 in the human genome. Approximately 2% of lung tumors harborROS1 fusions (Bergethon et al. 2012. J Clin Oncol. 2012 Mar. 10;30(8):863-70). The sequence of the human ROS1 gene and its structure areset forth in entry 6098 in NCBI's gene database; NCBI ReferenceSequence: NG_033929.1. See, e.g., Uguen et al Future Oncol. 201612:1911-28.

The target genes assessed for non-actionable sequence variations includeany cancer-related gene, including but not limited to AKT1, ALK, BRAF,CCND1, CDKN2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, GATA3,GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MET, MYC,NFE2L2, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, PPP2R1A, PTEN, ROS1, STK11,TP53 and U2AF1.

In some embodiments, the target genes assessed for non-actionablesequence variations include STK11 and KRAS. The non-actionable sequencevariations in STK11 include all single nucleotide variations and indelsthat result in a coding sequence or splice-site mutation. In someembodiments, a non-actionable sequence variation in STK11 may reduce orabolish the activity of STK11. The non-actionable sequence variations inKRAS include all single nucleotide variations and indels that result ina coding sequence or splice-site mutation and/or one or a combination ofhotspot variations selected from the group consisting of G12A, G12C,G12D, G12F, G12I, G12R, G12S, G12V, G13C, G13D, G13R, GQ60-61GK, Q61H,Q61L and Q61R. In some embodiments a non-actionable sequence variationin KRAS may activate KRAS.

In some embodiments, a non-actionable sequence variation may be mutuallyexclusive to an actionable sequence variation. For example, if a G12D(or other) variation in KRAS is identified, the cancer is unlikely to beassociated with an actionable sequence variation. Likewise, in thepresent study, it was found that non-actionable sequence mutations inSTK11 and KRAS are mutually exclusive with actionable sequencevariations in EGFR, ALK, ROS1 and BRAF. As such, if a non-actionablemutation in KRAS or STK11 is identified, then the patient may be treatedwith a non-targeted therapy such as radiation therapy, systemic or localchemotherapy, immunotherapy (e.g., immune checkpoint inhibition) orsurgery, without a targeted therapy. For non-small cell lung cancer, ifno actionable sequence variations are identified but a non-actionablemutation is found in KRAS or STK11, for example, then the patient may besubjected to chemotherapy using a platinum-based antineoplastic drugsuch as cisplatin, which may be used on its own or as a combinationtherapy with pemetrexed or gemcitabine.

In some embodiments, the allele frequency of any non-actionable sequencevariation identified in (a) is used to predict the probability ofdetecting or missing an actionable variant in the same sample. Insequencing a PCR product that contains a sequence variation that ispresent in a minority of the molecules, some of the sequence reads willbe from the variant molecules while others will not (e.g., will be fromthe “wild type” sequence). The “allele frequency”, i.e., frequency ofreads that are from the variant molecules can be calculated by, forexample, dividing the number of reads from the variant molecules by thetotal number of reads.

In these embodiments, the method may comprise predicting whether anactionable variant has been missed in a test sample, where thepredicting is based on the allele frequency of any non-actionablesequence variation identified and the distribution of allele frequenciesin actionable and non-actionable genes in reference samples and,optionally, the calculated sensitivity of the sequencing assay atdifferent allele frequencies. In other words, whether an actionablemutation has been missed in the cfDNA can be determined by examining thedistribution of allele frequencies of sequence variations, actionableand non-actionable, in a training set of reference samples with knownmutation profiles. In these embodiments, the prediction process maycomprise: i) comparing the distribution of allele frequencies of eachactionable sequence variation, gene containing multiple variants orgroup of genes containing multiple variants to each non-actionablevariant, gene containing non-actionable variants or group of genescontaining non-actionable sequence variations either in tumour materialfrom reference patients with the same cancer or previous cfDNA testsfrom patients with the same cancer, ii) comparing the allele frequencyof the non-actionable variant detected in the test sample to (i) inorder to predict the likely allele frequency or distribution of possibleallele frequencies of any actionable variants if present; and iii)comparing the likely allele frequency or distribution of possible allelefrequencies of actionable variants if present to the sensitivity of theassay at different levels in order to predict if an actionable variantis either not present or could be present but missed. In theseembodiments, the distributions of allele frequencies in actionable andnon-actionable genes may be compared using a linear regression model.

In some embodiments, the method may comprise analyzing white blood cellDNA from the patient and determining whether any of the actionable ornon-actionable sequence variations identified in step (a) are due tohematopoiesis of indeterminate potential or a germ-line variation andcan be eliminated from step (b). In these embodiments, the method mayinvolve comparing the genetic variations called using cfDNA to thegenetic variations called using the white blood cell DNA. If a variationis identified in both samples, then it may be identified as being morelikely to be a germ line variation or a somatic change in hematopoieticstem cells or other blood cells as opposed to a somatic mutation inctDNA. This embodiment provides a way to identify variations that may bepotentially due to clonal hematopoiesis of indeterminate potential(CHIP) (see, generally, Funari et al, Blood 2016 128:3176 and Heuser etal, Dtsch Arztebl Int. 2016 113: 317-322), or may be germ line variantsfor example.

An alternative embodiment may be used for the treatment of colorectalcancer patient without the need for a tissue biopsy. In this embodiment,the method may comprise: (a) performing or having performed a sequencingassay on cell-free DNA (cfDNA) from a sample of blood from the patientto determine if the cell-free DNA comprises i. one or more RAS mutationsin either NRAS or KRAS and ii. non-actionable sequence variations in oneor more target genes, and (b) treating the patient using the followingmethod: i. administering a therapy suitable for the treatment ofcolorectal cancer that contains RAS mutations if the patient isidentified as having a RAS sequence variation (e.g., FOLFIRI, FOLFOX orCAPEOX ±bevacizumab), and ii. administering an anti-epidermal growthfactor receptor (EGFR) therapy (e.g., a therapy that comprises ananti-EGFR therapy such as cetuximab or panitumumab) ±chemotherapy (e.g.,FOLFIRI or FOLFOX) to the patient in the absence of any follow-upgenetic testing on DNA extracted from a tissue biopsy if one or morenon-actionable sequence variations and no RAS mutations are identified.In colorectal cancer KRAS is mutated in approximately 40% of casesmostly in exon 2 codons 12 (70-80%) and 13 (15-20%). The remainingmutations are mainly located in exon 3 codons 59-61 and in exon 4, whichincludes codons 117 and 146. Mutations in NRAS are present inapproximately 3% to 5% of colorectal cancer samples particularly in exon3 codon 61 (60%) and in exon 2 codons 12, 13. In these embodiments, thetarget genes assessed for non-actionable sequence variations maycomprise APC and TP53. Like for the prior embodiments, the allelefrequency of any non-actionable sequence variation identified in (a) canbe used to predict the probability of detecting or missing a RASmutation in the sample. In some embodiments the BRAF gene may also beanalysed and in the absence of a BRAF V600E mutation being detected anynon-actionable sequence variations identified may be used to determinethe likelihood of one being missed as patients with such mutations arealso unlikely to respond to anti-epidermal growth factor receptor (EGFR)therapy.

In some embodiments, the method may comprise predicting whether a RASmutation has been missed in a test sample, wherein the predicting isbased on the distribution of allele frequencies for the one or more RASmutations and non-actionable variants in reference samples and,optionally, the calculated sensitivity of the sequencing assay atdifferent allele frequencies. In these embodiments, the predicting maycomprise i) comparing the distribution of allele frequencies of each RASmutation to each non-actionable variant, gene containing non-actionablevariants or group of genes containing non-actionable sequence variationseither in tumour material from reference patients with the same canceror previous cfDNA tests from patients with the same cancer, ii)comparing the allele frequency of the non-actionable variant detected inthe test sample to (i) in order to predict the likely allele frequencyor distribution of possible allele frequencies of the one or more RASmutations, if present; and iii) comparing the likely allele frequency ordistribution of possible allele frequencies of the one or more RASmutations, if present, to the sensitivity of the assay at differentlevels in order to predict if a RAS mutation is either not present orcould be present but missed. In any embodiment, the distributions ofallele frequencies in the one or more RAS mutations and non-actionablevariants can be compared using a linear regression model.

This alternative method may also involve analyzing white blood cell DNAfrom the patient and determining whether any of the RAS mutations ornon-actionable sequence variations identified in step (a) are due tohematopoiesis of indeterminate potential or a germ-line variation andcan be eliminated from step (b).

Exemplary implementations of the methods described above areschematically illustrated in the flow charts shown in FIGS. 1-4.

FIG. 1, flowchart A, illustrates the conventional way for makingtreatment decisions using cfDNA. In the conventional method, if noactionable changes are detected in cfDNA, then a tissue biopsy will beobtained and analyzed. FIG. 2, flowchart B, illustrates some of theprinciples of the present method. In these embodiments, if no actionablechanges are detected in cfDNA, then the non-actionable changes in thecfDNA can be examined to determine if an actionable change was missed.If analysis of the non-actionable changes indicates that actionablechanges are unlikely, then the patient can be treated in the absence ofa tissue biopsy from the patient.

FIG. 2 illustrates one way in which the method of flowchart A of FIG. 1can be applied to make treatment decision for non-small cell lung cancer(NSCLC). In this method, if there are no actionable changes in EGFR,ALK, ROS1 and BRAF, but there are non-actionable changes in KRAS orSTK11, then the patient can be treated in the absence of a tissue biopsyfrom the patient.

FIG. 3 illustrates one way in which treatment decisions can be madeusing allele frequencies. In this method, if there are no actionablechanges in EGFR, ALK, ROS1 and BRAF, the allele frequency ofnon-actionable changes can be used to determine the likelihood ofwhether an actionable change in those genes has been missed.

FIG. 4 illustrates one way in which treatment decisions can be made incolorectal cancer (CRC). By analysing non-actionable changes in genesincluding TP53 and APC, one can predict which patients are unlikely tohave a RAS variant and which patients have too little ctDNA to determineif a RAS variant is present and therefore need further tissue testing.

In many embodiments, the method may comprise sequencing at least part ofthe coding sequences of several genes (e.g., EGFR, BRAF, KRAS and STK11)as well as at least part of ALK and ROS1 in a sample of the cfDNA.Methods for sequencing target sequences in cfDNA are known and, in someembodiments, the method may comprise enriching for or amplifying targetsequences by PCR prior to sequencing (see, e.g., Forshew et al, Sci.Transl. Med. 2012 4:136ra68, Gale et al, PLoS One 2018 13:e0194630 andWeaver et al, Nat. Genet. 2014 46:837-843, among many others). ALK andROS1 fusions may be identified using similar methods, e.g., using PCRand the sequencing the products. These method may make use of primerpairs in which one primer hybridizes to the ALK or ROS1 gene and anotherprimer hybridizes to a gene encoding a potential fusion partner for ALKor ROS1. In some embodiments, the method does not involve shotgunsequencing an unenriched/unamplified sample, or sequencing the entireexome. Rather, the sequencing may be done as part of a larger sequencingeffort that targets at least part of the coding sequences for up to 500,e.g., up to 100 or up to 50 genes, focusing on the coding sequences andfusions of the genes of interest. In some embodiments this targeting maybe performed by hybrid capture.

After the sequences have undergone initial processing, the sequences areanalyzed to identify sequence variations. This may be done by comparingthe test sequence to a reference sequence, for each sequence beinganalyzed, and the identifying positions that contain a change in anucleotide. In some cases, this may comprise calling mutations de novo(e.g., using the method described by Forshew, supra, or another suitablemethod) and then determining which of those mutations are actionable ornon-actionable. Calling sequence variations in cell-free DNA can bechallenging because the variant sequences are generally in the minority(e.g., less than 10% of the sequence). As such, if an ampliconsequencing strategy is employed, the method may comprise: (a) for eachnucleotide position of a particular amplicon, determining, e.g.,plotting, an error distribution that shows how often amplificationand/or sequencing errors occur at different sequencing depths; (b) basedon the distribution for each position of the sequence, determining athreshold frequency for each different sequencing depth at or abovewhich a true genetic variation can be detected; (c) sequencing thesample to obtain plurality of reads for an amplicon; and determining,for each position of the amplicon, whether the frequency of a potentialsequence variation in the sequence reads is above or below thethreshold. Mutation may be identified (or “called”) at a position if thefrequency of sequence reads that contain the variation is above thethreshold. In some cases, a substitution may be identified only if itoccurs in the same amplicon from multiple independent amplificationreactions. As would be apparent, if the sequencing is done using anamplicon approach, the method may comprise amplifying the codingsequences of the genes in a multiplex PCR reaction in which at least 10amplicons (e.g., more than 10 and less than 50,000 amplicons, more than10 and less than 10,000, more than 10 and less than 5,000 amplicons,more than 10 and less than 1,000 amplicons or more than 10 and less than500 amplicons) or more than 10 and less than 100 amplicons are amplified(in duplicate, triplicate or quadruplicate, for example) and sequencingthe amplicons. More or less amplicons can also be sequenced, if needed.In some embodiments, the primers used for amplification may not becompletely specific for a single sequence, which can allow severalhundred or several thousand amplicons to be consistently amplified in asingle reaction. The amplicons sequenced can be of any suitable lengthand may vary in length. In some embodiments, the length of eachamplicon, independently, may be in the range of 50 bp to 500 bp,although longer or shorter amplicons may be used in someimplementations.

Next, the sequence variations are identified, the sequence variationsmay be classified as actionable or non-actionable. In some embodiments,the method may further comprise sequencing at least part of the codingsequences of KRAS and STK11 in the sample of cfDNA. In theseembodiments, the method may involve analyzing the sequences to determineif there are any activating mutations in KRAS and loss of functionmutations in STK11. Examples of loss of function mutations include, butare not limited to mutations that generate a stop codon, mutations atsplice junctions, and mutations that substitute a critical amino acidfor another.

A patient that, based on the analysis of the patient's cell-free DNA,appears to be have no actionable mutations in EGFR, ALK, ROS1 or BRAFbut a non-actionable mutation in KRAS or STK11 can be treated with anon-targeted therapy without a tissue biopsy.

The sequencing step may be done using any convenient next generationsequencing method and may result in at least 10,000, at least 50,000, atleast 100,000, at least 500,000, at least 1M at least 10M at least 100Mor at least 1B sequence reads. In some cases, the reads are paired-endreads. As would be apparent, the primers used for amplification may becompatible with use in any next generation sequencing platform in whichprimer extension is used, e.g., Illumina's reversible terminator method,Roche's pyrosequencing method (454), Life Technologies' sequencing byligation (the SOLiD platform), Life Technologies' Ion Torrent platform,QIAGEN's GeneReader platform or Pacific Biosciences' fluorescentbase-cleavage method. Examples of such methods are described in thefollowing references: Margulies et al (Nature 2005 437: 376-80); Ronaghiet al (Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005309: 1728); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al(Methods Mol Biol. 2009; 553:79-108); Appleby et al (Methods Mol Biol.2009; 513:19-39) English (PLoS One. 2012 7: e47768) and Morozova(Genomics. 2008 92:255-64), which are incorporated by reference for thegeneral descriptions of the methods and the particular steps of themethods, including all starting products, reagents, and final productsfor each of the steps. Nanopore sequencing could be employed in certaincases.

In some embodiments, the method may comprise providing a reportindicating whether there are any: i. actionable sequence variations,e.g., in EGFR, ALK, ROS or BRAF and ii. non-actionable sequencevariations, e.g., in KRAS or STK11. In addition, a report may provideoptions for approved (e.g., FDA approved) therapies if an actionablesequence variation is identified (e.g., a list of targeted therapies).If no actionable sequence variations and one or more non-actionablesequence variations are identified, then the report may: i. provideoptions for approved (e.g., FDA approved) non-targeted therapies for thecancer and ii. a statement that indicates that analysis of a tissuebiopsy is unnecessary before treatment. In some embodiments the reportmay provide levels of confidence that specific actionable variants arenot present.

In some embodiments, the report may be in an electronic form, and themethod comprises forwarding the report to a remote location, e.g., to adoctor or other medical professional to help identify a suitable courseof action, e.g., to identify a suitable therapy for the subject. Thereport may be used along with other metrics to determine whether thesubject may be susceptible to immune checkpoint inhibition.

In any embodiment, a report can be forwarded to a “remote location”,where “remote location,” means a location other than the location atwhich the sequences are analyzed. For example, a remote location couldbe another location (e.g., office, lab, etc.) in the same city, anotherlocation in a different city, another location in a different state,another location in a different country, etc. As such, when one item isindicated as being “remote” from another, what is meant is that the twoitems can be in the same room but separated, or at least in differentrooms or different buildings, and can be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” informationreferences transmitting the data representing that information aselectrical signals over a suitable communication channel (e.g., aprivate or public network). “Forwarding” an item refers to any means ofgetting that item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. Examples of communicatingmedia include radio or infra-red transmission channels as well as anetwork connection to another computer or networked device, and theinternet, including email transmissions and information recorded onwebsites and the like. In certain embodiments, the report may beanalyzed by an MD or other qualified medical professional, and a reportbased on the results of the analysis of the sequences may be forwardedto the patient from which the sample was obtained.

In computer-related embodiments, a system may include a computercontaining a processor, a storage component (i.e., memory), a displaycomponent, and other components typically present in general purposecomputers. The storage component stores information accessible by theprocessor, including instructions that may be executed by the processorand data that may be retrieved, manipulated or stored by the processor.

The storage component includes instructions for providing a score usingthe measurements described above as inputs. The computer processor iscoupled to the storage component and configured to execute theinstructions stored in the storage component in order to receive patientdata and analyze patient data according to one or more algorithms. Thedisplay component may display information regarding the diagnosis of thepatient.

The storage component may be of any type capable of storing informationaccessible by the processor, such as a hard-drive, memory card, ROM,RAM, DVD, CD-ROM, USB Flash drive, write-capable, and read-onlymemories. The processor may be any well-known processor, such asprocessors from Intel Corporation. Alternatively, the processor may be adedicated controller such as an ASIC.

The instructions may be any set of instructions to be executed directly(such as machine code) or indirectly (such as scripts) by the processor.In that regard, the terms “instructions,” “steps” and “programs” may beused interchangeably herein. The instructions may be stored in objectcode form for direct processing by the processor, or in any othercomputer language including scripts or collections of independent sourcecode modules that are interpreted on demand or compiled in advance.

Data may be retrieved, stored or modified by the processor in accordancewith the instructions. For instance, although the diagnostic system isnot limited by any particular data structure, the data may be stored incomputer registers, in a relational database as a table having aplurality of different fields and records, XML documents, or flat files.The data may also be formatted in any computer-readable format such as,but not limited to, binary values, ASCII or Unicode. Moreover, the datamay comprise any information sufficient to identify the relevantinformation, such as numbers, descriptive text, proprietary codes,pointers, references to data stored in other memories (including othernetwork locations) or information which is used by a function tocalculate the relevant data.

EXAMPLES

Aspects of the present teachings can be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Methods

Patients with advanced non-squamous NSCLC were recruited in 2prospective US clinical studies (INI-001 [NCT02906852], GRN-ALV[NCT03116633]). Samples from 10 patients were also obtained from acommercial biobank [Asterand, US].

Patients were included if they met the following criteria: writteninformed consent; aged 18, stage IIIB/IV NSCLC, had not received therapyfor advanced NSCLC; blood for plasma ctDNA analysis collected within 12weeks of NSCLC tissue biopsy; no anti-cancer therapy between the tissueand plasma collection.

The primary aim was to examine concordance of ctDNA and tissueprofiling. All patients meeting the above criteria were eligibleregardless of tissue availability to allow the comparison of ctDNAprofiles in patients with and without tissue for profiling.

All studies were undertaken within recognized ethical principles laiddown in ICH GCP and the declaration of Helsinki and were subject toIRB/Ethical review and approval.

ctDNA, tissue analysis and concordance analysis: Blood was collectedinto Streck-DNA tubes (Streck Inc, US) and shipped to the Inivata CLIAaccredited laboratory (Morrisville, US) for InVision ctDNA analysis.Full assay details have been described previously [9, 10]. Briefly,blood was processed to plasma by centrifugation. Following plasmaextraction, plasma was stored at −80° C. according to validatedspecifications until analysis in batch. Cell free DNA (cfDNA) wasextracted using the QIAamp Circulating Nucleic Acid kit (Qiagen).Following quality control, sequencing libraries were prepared using atwo-step amplification process, and libraries were sequenced by IlluminaNextSeq 500. Sequencing data were analyzed using the Inivata analyticalpipeline to identify genomic alterations.

Where sufficient tissue was available, CGP was performed in aCLIA-certified laboratory (Caris Life Science, US). Direct sequenceanalysis was performed on genomic DNA isolated from formalin-fixedparaffin-embedded (FFPE) tumor samples using the Illumina NextSeqplatform. An Agilent custom-designed SureSelect XT assay was used toenrich 592 whole-gene targets and all variants reported were detectedwith >99% confidence. Fusion analysis was performed using the ArcherFusionPlex Solid Tumor Panel and the Illumina MiSeq. When patients hadinsufficient tissue for CGP, tissue testing was allowed as per thetreating institutions routine pathways. Where both central CGP and localdata was available, the centralized CGP data was utilized forconcordance analysis.

Both InVisionFirst and tissue analysis were performed blinded from eachother. Calls made in either ctDNA or tissue in genomic regions that werenot covered by testing in the other were excluded from concordanceanalysis. The calling nomenclature for all identified mutations wasreviewed along with underlying sequencing data where present to ensurethat mutations were named consistently and all calls were correctlyclassified for concordance.

ddPCR validation data: Thirty-three patients from the INI-001 study alsounderwent plasma ddPCR testing for key genomic alterations (KRASG12C/G12D/G12V, EGFR exon19del/T790M/L858R, BRAF V600E, ALK/ROS1fusions) via a commercial assay provider (GeneStrat, Biodesix Inc, US)as part of their routine standard of care. Blood samples were collectedand shipped according to the standard specifications. Analysis wascompleted without any knowledge of tissue or ctDNA testing results.

Statistical analysis: All analyses were performed using R version 3.2.5.In the concordance analysis, the data was summarized using a 2 by 2table, referring to tissue as the standard:

Tissue positive Tissue negative Plasma positive True positive (TP) Falsepositive (FP) Plasma False negative True Negative negative (FN) (TN)

The following definitions were used:

-   -   Sensitivity=TP/(TP+FN)    -   Specificity=TN/(TN+FP)    -   PPV=TP/(TP+FP)    -   NPV=TN/(TN+FN)

Results

A total of 264 eligible patients were recruited across 41 centers.Baseline demographics for the cohort are shown in Table 2 and wereconsistent with expectations. Patients with and without tissue testinghad similar demographics. A summary of some of the results is shown inFIG. 5.

TABLE 2 Cohort Demographics Patients without Patients with tissue tissuefor All for testing testing patients n 86 178 264 Age (mean (sd)) 68.2(10.9) 66.6 (11.1) 67.1 (11.0) Smoking status (%) Current smoker 22.131.5 28.4 Former smoker 60.5 55.1 56.8 Never smoked 17.4 12.9 14.4Missing 0.0 0.6 0.4 Race (%) American indian or Alaska native 0.0 0.60.4 Asian 3.5 1.7 2.3 Black or African American 7.0 11.2 9.8 White 84.986.0 85.6 Other 4.7 0.6 1.9 Histology (%) Adenocarcinoma 94.2 96.1 95.5Large cell carcinoma 1.2 0.0 0.4 Neuroendocrine carcinoma 0.0 0.6 0.4Sarcomatoid 1.2 0.0 0.4 Missing 3.5 3.4 3.4 BMI (mean (sd)) 27.1 (6.0)26.4 (6.1) 26.6 (6.1) Sex = M (%) 51.2 47.2 48.5 Cancer stage (%) 3B10.5 16.9 14.8 4 88.4 79.2 82.2 Missing* 1.2 3.9 3.0 *All patientsincluded were confirmed as eligible based on TNM staging

InVisionFirst ctDNA Profile

All patients were successfully tested for SNVs, indels andamplifications. Testing for ALK/ROS1 fusions was successful in 252patients (95.5% of patients). Overall, 204 (77.3%) patients had one ormore alterations detected by ctDNA. The mean number of alterationsidentified per patient was 1.5. Of the SNVs and indels identified, 35.5%had an allele fraction lower than 1%, and 23.1% had an allele fractionlower than 0.5%.

The predominant alterations identified were TP53 (47% patients) and KRAS(32% patients). Twenty-seven SNVs or indels in EGFR exon 18-21 wereidentified in 26 patients (10%). Gene fusions were identified in 5patients (2%), including EML4-ALK in 4 patients and CD74-ROS1 in 1patient. The pattern and frequency of genomic alterations was similaracross patients with and without tissue.

Tissue Testing and Tissue-ctDNA Concordance

Of the 264 recruited patients, 178 had successful tissue testing for atleast 1 genomic alteration. One hundred and sixty five patients (62.5%)were tested for any point mutation/indel, and 159 (60.2%), were testedfor ROS1 and/or ALK fusions. The most frequently tested gene in tissuewas EGFR (164 patients, 62.1%). A total of 95 patients were tested forall 8 of the key genes (ERBB2, ALK, ROS1, BRAF, MET, EGFR, STK11 andKRAS), and 121 were tested for fusions in ALK and ROS1 and mutations inEGFR, MET and BRAF.

Considering tissue as the reference, the sensitivity of InVisionFirstacross the entire panel was 70.6%. Considering only clinicallyactionable alterations in 8 genes of most relevance, the sensitivity was73.9% with a PPV of 97.8% (Table 3). The PPV was 100% when onlyconsidering the directly actionable variants (ALK/ROS1 fusions/EGFRexons18-21/ERBB2 insertions/MET exon14 splice/BRAF V600E).

TABLE 3 Summary of tissue concordance data. Tissue and Tissue Plasma NoPlasma only only call PPV NPV Sensitivity Specificity ALK/ROSI 2 3 0 292100.0 99.0 40.0 100.0 fusions BRAF V600E 5 2 0 140 100.0 98.6 71.4 100.0EGFR (exons 13 5 0 146 100.0 96.7 72.2 100.0 18-21) ERBB2 exon 2 0 0 137100.0 100.0 100.0 100.0 20 ins KRAS 48 12 1 86 98.0 87.8 80.0 98.9 METexon 14 3 3 0 133 100.0 97.8 50.0 100.0 splice STK11 15 6 1 93 93.8 93.971.4 98.9 Key 8 genes* 88 31 2 1027 97.8 97.1 73.9 99.8 All Genes 156 6532 4135 83.0 98.5 70.6 99.2 *Key 8 genes refers to the combination ofall directly actionable mutations (ALK/ROS1 fusions, BRAF V600E, EGFRexons 18-21, ERBB2 insertions, MET exon 14 splice) and KRAS and STK11variants.

InVisionFirst detected 32 mutations in 23 patients that were notdetected by tissue analysis, including TP53 (17 mutations), PIK3CA (3),NRAS (3) and ERBB2 (2). For 30 of these, read alignment data from tissueNGS was available. Review by the testing laboratory found evidence ofthe mutations below the standard calling threshold in 6 of the 30mutations: PIK3CA E542K (3 occurrences), KRAS G12C, MET D1249N, and TP53V197M.

Two hundred and four patients (77.27%) had at least one mutationdetected by InVisionFirst. In this cohort, the sensitivity was 88.0% forclinically relevant alterations in the key 8 genes.

Utility Analysis

Tissue CGP was performed for all patients where sufficient tissue wasavailable and was funded as part of the study. The tissue testingundertaken across the study is therefore considered representative ofthe real-world potential utility of tissue testing within thispopulation. Despite this, InVisionFirst testing resulted in a muchhigher rate of testing compared to tissue testing across the entirerecruited population. Table 4 details the most clinically relevantalterations detected across all patients enrolled. A total of 48patients qualified for a targeted treatment based on InVisionFirsttesting, and 38 patients based on tissue testing. 48% of the actionablealterations detected by InVisionFirst were in patients who had not beentested for that alteration in tissue due to incomplete tissue testingdespite funding by the study.

TABLE 4 Summary of actionable and rule-out status using the liquidbiopsy data. Plasma Plasma Tissue Tissue Class Subclass (n) (%) (n) (%)Total 264 100.00 264 100.00 Actionable 48 18.18 38 14.39 EGFR exons18-21 26 9.85 18 6.82 ALK-ROS1 fusions 5 1.89 5 1.89 ERBB2 exon 20 41.52 2 0.76 insertions BRAF V600E 6 2.27 7 2.65 MET exon 14 7 2.65 62.27 splice KRAS/STK11 94 35.61 70 26.52 and no actionable mutations

Mutations in KRAS and STK11 are generally mutually exclusive withactionable driver mutations in untreated non-squamous NSCLC [11-13] andtheir detection could provide additional confidence that patientswithout actionable alterations are true-negative rather thanfalse-negatives. Combining patients for whom InVisionFirst identified anactionable alteration (18.2% of the cohort) with patients where it didnot identify those but identified KRAS and/or STK11 mutations, a totalof 142 patients (53.8% of the cohort) had an actionable alterationdetected or ruled out. Of the KRAS/STK11 mutations detected in plasma inthese patients, 90 had the same variant tested in tissue and 88 of thesewere detected (97.8% PPV, Table 3). The two mutations not detected intissue included a KRAS G12C which was observed below the threshold forcalling and a mutation in STK11 detected in plasma in a patient who wasalso KRAS positive by both tissue and plasma. In the 96 patients whereInVisionFirst identified mutations in KRAS and/or STK11, tissue dataavailable did not detect any actionable alterations.

Orthogonal Validation by ddPCR

Plasma orthogonal testing by ddPCR revealed an overall concordance ofalteration calls of 98.8% (330/334) with PPV of 95.7% and NPV of 99.1%when considering ddPCR as the reference. Discordance was observed in 4alterations: EGFR exon19del (1 case) and KRAS G12C (2 cases) weredetected by InVisionFirst but not by ddPCR. In one case EGFR L858R wasdetected by ddPCR but not by InVisionFirst. Tissue was available in twoof these cases and confirmed the presence of one KRAS G12C mutation andthe EGFR L858R mutations. Lastly, one KRAS G12A mutation was detected byboth the InVisionFirst assay and tissue sequencing but was identified asKRAS G12D by the ddPCR assay.

DISCUSSION

The study described above is believed to be the first prospectivevalidation of a ctDNA NGS platform for molecular stratification ofpatients with advanced untreated NSCLC.

Using tissue as the reference, concordance for the full 36 genes in theInVisionFirst panel with matched tissue profiling was 97.8%. Consideringclinically actionable alterations in 8 genes that can most influenceroutine clinical patient management, the PPV was 97.8%, 97.1% NPV, 73.9%sensitivity and 99.4% specificity. Of all mutations detected in plasma,23% had an allele fraction below 0.5%, highlighting the need for highlysensitive assays with strong performance at low allelic frequencies. TheInVisionFirst assay has demonstrated excellent sensitivity in analyticalvalidation studies [9], but despite this high level of sensitivity,approximately 23% of these newly diagnosed stage IIIb/IV NSCLC patientshad no mutations detected in ctDNA.

High sensitivity should be coupled with high specificity to ensure thatfalse positive results do not lead to inappropriate therapy. Across thefull panel, the PPV was 83.0%, compared to 100% for actionable driveralterations only, the difference being a consequence of 32non-actionable variants detected in plasma but not in tissue. In 6 ofthese cases, there was evidence for the variant below thresholdsrequired for calling in tissue. 16 of the remaining 26 calls were TP53variants. These may be sub-clonal events that only occur at low levelsor may be completely absent from the biopsy site. Theover-representation of TP53 in ctDNA may also be explained by clonalhematopoiesis [14]. Of note, where tissue was available, all clinicallyactionable alterations detected by InVisionFirst in plasma wereconfirmed by tissue profiling. This provides reassurance of the highspecificity of the assay and is supported by previous studies ofInVisionFirst in NSCLC [15, 16].

In total, 18.2% of patients tested by InVisionFirst had an actionablechange detected. An additional 35.6% were found to have a genomicalteration generally mutually exclusive with such actionablealterations. 53.8% of patients therefore had an informative result thatcould prevent the need for additional invasive biopsies. The strength ofthis ‘rule out’ classification was confirmed by the absence of anyactionable alterations detected in available tissue in these patients.

Despite excitement regarding ctDNA NGS platforms, there are currently norobust studies in an equivalent clinical setting to provide comparisonsacross assays. Since ctDNA levels vary between patients at differentstages of disease [7], sensitivity is affected by the population in thestudy. Compared to newly diagnosed patients studied here, the clinicalsensitivity of InVisionFirst in previous studies was higher in therelapse setting, with 100% sensitivity (compared to tissue) reported forthe EGFR driver mutation at relapse in 30 TKI treated NSCLC patients[16]. Another assay was also reported to have sensitivities ranging from35.7% to 90.3% in different disease settings [17-19]. Taken together,these observations dictate that clinical validation of assays should beperformed in unselected patients from the intended use population withclinical characteristics consistent with the proposed clinicalindication prior to clinical adoption [20].

The clinical sensitivity of the InVisionFirst assay demonstrated here iscomparable to published data on the FDA approved Roche CobasV2single-gene EGFR ctDNA assay [21]. Such single-gene tests only identifythe small subset of patients with mutations in those genes and areinconclusive for the great majority of patients who potentially requireadditional testing. The InVisionFirst assay provides data across a panelof genes and can provide a definitive result in >50% of patients througha rule-in/rule-out approach.

Tissue testing for the most common alterations was only successful in62% of patients in the study, consistent with statistics reported in arecent study across community oncology institutions [3]. Full CGP wassuccessful in significantly fewer patients. Routine implementation ofctDNA testing by InVisionFirst could help to increase the proportion ofpatients eligible for targeted therapies. Within this study,InVisionFirst identified 48 actionable alterations compared to 38 thatwere detected by standard of care tissue testing supplemented by CGP.Nearly half of the alterations detected by InVisionFirst were inpatients who were not profiled for the alteration due to limitations intissue testing. This increased detection of actionable alterations wouldbe delivered while reducing costs, patient discomfort and complicationsassociated with repeated invasive tissue biopsies.

Patients with advanced NSCLC progress rapidly and the time taken toobtain results of molecular profiling is therefore paramount. Resultsfor the InVisionFirst assay are now routinely available in 7-days fromblood draw. Utilization of such testing early in the work-up of patientswith advanced NSCLC may therefore enable earlier therapeuticintervention.

REFERENCES

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It will also be recognized by those skilled in the art that, while theinvention has been described above in terms of preferred embodiments, itis not limited thereto. Various features and aspects of the abovedescribed invention may be used individually or jointly. Further,although the invention has been described in the context of itsimplementation in a particular environment, and for particularapplications (e.g. ctDNA analysis) those skilled in the art willrecognize that its usefulness is not limited thereto and that thepresent invention can be beneficially utilized in any number ofenvironments and implementations where it is desirable to examine cfDNA,Accordingly, the claims set forth below should be construed in view ofthe full breadth and spirit of the invention as disclosed herein.

1-21. (canceled)
 22. A method of treating a cancer patient, comprising:(a) performing or having performed a sequencing assay on cell-free DNA(cfDNA) from a biological sample from the patient, wherein thesequencing assay targets one or more RAS mutations and one or more othersequence variations associated with the patient's cancer; (b)identifying or having identified no RAS mutations and one or more othersequence variations associated with the patient's cancer; and (c)administering, based on the identification of no RAS mutations and theone or more other sequence variations associated with the patient'scancer, a therapy suitable for the treatment of cancer that does notcontain RAS mutations, wherein the decision to administer the therapy ismade without considering data obtained from a tumor biopsy.
 23. Themethod of claim 22, wherein the one or more RAS mutations are in theNRAS and/or KRAS genes.
 24. The method of claim 23, wherein the one ormore RAS mutations comprise mutations selected from the group consistingof the G12A, G12C, G12D, G12F, G12I, G12R, G12S, G12V, G13C, G13D, G13R,GQ60-61GK, Q61H, Q61L and Q61R mutations in the KRAS gene.
 25. Themethod of claim 22, wherein the method comprises: calculating the allelefrequencies of the one or more other sequence variations identified instep (b); and calculating the probability that there are RAS mutationsbased on the allele frequencies of the one or more other sequencevariations.
 26. The method of claim 22, wherein the one or more othersequence variations identified in (b) are APC and TP53.
 27. The methodof claim 22, wherein the therapy administered in (c) is ananti-epidermal growth factor (EGFR) therapy.
 28. The method of claim 27,wherein the anti-epidermal growth factor (EGFR) therapy comprisescetuximab or panitumumab.
 29. The method of claim 27, wherein the one ormore other sequence variations identified in (b) are activatingmutations in the EGFR gene.
 30. The method of claim 22, wherein thetherapy administered in (c) is a platinum-based chemotherapy.
 31. Themethod of claim 22, further comprising: analyzing white blood cell DNAfrom the patient; determining whether any of one or more other sequencevariations are due to hematopoiesis of indeterminate potential or agerm-line variation; and eliminating sequence variations that are due tohematopoiesis of indeterminate potential or a germ-line variation fromthe analysis of step (b).
 32. The method of claim 22, wherein thepatient has colorectal cancer.
 33. The method of claim 22, wherein thebiological sample is a blood sample.
 34. A method of treating a cancerpatient, the method comprising: (a) performing or having performed asequencing assay on cell-free DNA (cfDNA) from a biological sample fromthe patient, wherein the sequencing assay targets one or more RASmutations and one or more other sequence variations associated with thepatient's cancer; (b) identifying whether the cfDNA comprises one ormore RAS mutations, and one or more other sequence variations associatedwith the patient's cancer; and (c) treating the patient by: i.administering a therapy suitable for the treatment of cancer thatcontains RAS mutations if one or more RAS mutations are identified, orii. administering a therapy suitable for the treatment of cancer thatdoes not contain RAS mutations to the patient if no RAS mutations andone or more other sequence variations associated with the patient'scancer are identified; wherein the decision to administer the therapy ismade without considering data from a tumor biopsy.
 35. A method oftreating a cancer patient in the absence of data obtained from a tissuebiopsy, the method comprising: (a) performing a sequencing assay oncell-free DNA (cfDNA) from a sample of blood from the patient, whereinthe sequencing assay targets one or more RAS mutations and one or moreother sequence variations associated with the patient's cancer; (b)identifying, whether the cfDNA comprises i. one or more RAS mutations,and ii. one or more other sequence variations associated with thepatient's cancer; and (c) providing a report that indicates one of thefollowing options for treating the patient: i. administer a therapysuitable for the treatment of cancer that contains RAS mutations if oneor more RAS mutations are identified, and ii. administer a therapysuitable for the treatment of cancer that does not contain RAS mutationsto the patient if no RAS mutations and one or more other sequencevariations associated with the patient's cancer are identified.